CN111996250A - Molecular marker for developing gastric adenocarcinoma diagnosis and treatment product - Google Patents

Abstract

The invention discloses a molecular marker for developing diagnosis and treatment products for gastric adenocarcinoma, which is RP11-626H 12.2. The invention discovers that the expression level of RP11-626H12.2 is up-regulated in gastric adenocarcinoma patients for the first time, and through ROC curve analysis, RP11-626H12.2 is found to have a higher AUC value, which indicates that RP11-626H12.2 has higher accuracy and specificity when being applied to diagnosis of gastric adenocarcinoma.

Description

Molecular marker for developing gastric adenocarcinoma diagnosis and treatment product

Technical Field

The invention belongs to the field of biological medicines, relates to a molecular marker for developing gastric adenocarcinoma diagnosis and treatment products, and particularly relates to a molecular marker RP11-626H 12.2.

Background

Gastric cancer is one of the common malignant tumors in the world, has relatively poor prognosis and seriously threatens human health. China is a country with high incidence of gastric cancer, the incidence of new cases of gastric cancer accounts for more than 40% of new cases of stomach cancer every year, the death rate is the second, and the disease is a main disease seriously harming the health of Chinese residents. Because the proportion of the gastric cancer patients in the advanced stage of China is high, the 5-year survival rate is still lower than 30 percent even if the patients receive comprehensive treatment mainly by operation. In order to expand the treatment means of gastric cancer and improve the survival time of gastric cancer patients, researches on exploring the molecular mechanism of gastric cancer pathogenesis, searching biomarkers for diagnosis and prognosis and treatment targets are the problems to be solved at present.

Long non-coding RNA (1 ncRNA) refers to non-coding RNA with the length of more than 200 nucleotides, does not have an open reading frame and has no protein translation function. The 1ncRNA can be divided into 5 types according to the relationship with the nearest coding protein gene, (1) sense 1ncRNA, namely the same as the coding protein gene chain; (2) antisense 1ncRNA, i.e., different from the gene strand encoding the protein; (3) bidirectional 1ncRNA, namely, the 1ncRNA which is closest to the coding protein gene disperses or gathers in the direction of transcription; (4) intron 1ncRNA, i.e., located within the intron of the gene encoding the protein; (5) intergenic 1ncRNA, i.e., located in the 2 intergenic regions encoding proteins, has long been known as the "noise" of genome transcription, without biological function. The current increasing research results show that the 1ncRNA plays an important role in the process of regulating gene expression, and the important role in the occurrence, development, metastasis and recurrence processes of various tumors such as gastric cancer, gastric adenocarcinoma, colon cancer and liver cancer is also proved. Although some progress has been made, the research on the role of 1ncRNA in gastric cancer is still in the initial stage, the expression profile of 1ncRNA related to gastric cancer is unknown, and the specific role of 1ncRNA in the pathological mechanism of gastric cancer is less understood. The function of lncRNA in gastric cancer is deeply researched, important molecules for effectively regulating and controlling the occurrence and development of gastric cancer are found, and the method has important significance for guiding the research and development of clinical gastric cancer molecular diagnosis and molecular targeted drugs.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide lncRNA related to the occurrence and development of gastric cancer, thereby providing a molecular target for the diagnosis and treatment of gastric cancer and realizing the personalized diagnosis and treatment of patients.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a reagent capable of detecting the level of the RP11-626H12.2 gene in a sample.

Further, the reagent comprises:

a probe specifically recognizing RP11-626H 12.2; or

And (3) primers for specifically amplifying RP11-626H 12.2.

Furthermore, the primer sequence of the specific amplification RP11-626H12.2 gene is shown in SEQ ID NO. 1-2.

According to a second aspect of the invention there is provided a kit comprising a reagent according to the first aspect of the invention, said reagent being capable of detecting the level of the RP11-626H12.2 gene.

In a third aspect, the invention provides a chip comprising the reagent according to the first aspect of the invention, wherein the reagent is capable of detecting the level of the RP11-626H12.2 gene.

In a fourth aspect, the invention provides a strip comprising a reagent according to the first aspect of the invention, said reagent being capable of detecting the level of the RP11-626H12.2 gene.

In a fifth aspect, the invention provides a pharmaceutical composition comprising an effective amount of an inhibitor of RP11-626H 12.2.

Further, the inhibitor is an agent for reducing the expression level of RP11-626H 12.2. The inhibitor is selected from: an interfering molecule which uses RP11-626H12.2 or a transcript thereof as a target sequence and can inhibit the expression or gene transcription of RP11-626H12.2 gene, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. Preferably, the inhibitor is siRNA.

Furthermore, the composition also comprises other medicines compatible with the inhibitor and a pharmaceutically acceptable carrier and/or auxiliary material.

The medicaments of the invention can also be combined with other medicaments for the treatment of gastric cancer, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

A sixth aspect of the present invention provides a method of screening a candidate substance for treating gastric cancer, the method comprising:

treating a culture system expressing or containing an RP11-626H12.2 gene with a substance to be screened; and

detecting the level of RP11-626H12.2 gene in the system;

wherein, if the substance to be screened can inhibit the level of the RP11-626H12.2 gene, the substance to be screened is a candidate substance for treating gastric cancer.

A seventh aspect of the invention provides a use as claimed in any one of:

a. the use of a reagent according to the first aspect of the invention in the manufacture of a product for the diagnosis of gastric adenocarcinoma;

b. the use of a kit according to the second aspect of the invention in the manufacture of a product for diagnosing gastric adenocarcinoma;

c. the application of the chip of the third aspect of the invention in preparing a product for diagnosing gastric adenocarcinoma;

d. the application of the test paper of the fourth aspect of the invention in preparing a product for diagnosing gastric adenocarcinoma;

application of RP11-626H12.2 in constructing a calculation model for predicting gastric adenocarcinoma;

f. the application of the pharmaceutical composition of the fifth aspect of the invention in preparing a product for treating gastric adenocarcinoma.

The invention has the advantages and beneficial effects that:

the invention discovers that the differential expression of RP11-626H12.2 is related to the occurrence and development of gastric adenocarcinoma for the first time, and whether a subject suffers from gastric adenocarcinoma can be judged by detecting the expression level of RP11-626H12.2, thereby providing a molecular target for diagnosis and treatment of gastric adenocarcinoma.

Drawings

FIG. 1 is a ROC plot of RP11-626H12.2 as the sensed variable.

FIG. 2 is a graph showing the detection of the expression of RP11-626H12.2 gene in gastric cancer tissues by QPCR.

Detailed Description

According to the invention, through extensive and intensive research, the TCGA database is deeply mined to obtain the high-throughput data of gastric adenocarcinoma, the lncRNA which is key in the development of gastric adenocarcinoma is screened out by using a machine learning algorithm, the development mechanism of gastric adenocarcinoma is deeply known, the potential lncRNA which can be used for the diagnosis of gastric adenocarcinoma is screened out, and meanwhile, the relevant lncRNA is further verified to search for a real molecular marker which can be used for the diagnosis and treatment of gastric adenocarcinoma. According to the invention, through bioinformatics analysis, the expression up-regulation of RP11-626H12.2 in gastric adenocarcinoma is found for the first time, and QPCR experiments further prove that the expression level of RP11-626H12.2 in gastric adenocarcinoma is obviously higher than that of tissues beside cancer, the discovery of RP11-626H12.2 further enriches the research on pathogenesis of gastric adenocarcinoma, and simultaneously provides a new molecular means for diagnosis and treatment of gastric adenocarcinoma.

RP11-626H12.2 gene

RP11-626H12.2 is located on human No. 11 stain, and RP11-626H12.2 in the invention includes wild type, mutant or fragment thereof. Currently, two transcripts exist for RP11-626H12.2, and the transcript IDs are: ENST00000528507.1 and ENST 00000533170.1. A representative RP11-626H12.2 gene sequence is shown in ENST 00000528507.1.

It is known to those skilled in the art that when performing bioinformatic analysis on the original sequencing result, the sequencing result is usually aligned with a known gene, and the expression of the gene can be regarded as long as the sequenced fragment can be aligned with the related gene, so that when referring to a differentially expressed gene, different transcripts of the gene are also included in the present invention.

It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level. The present invention may utilize any method known in the art for determining gene expression.

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a matrix gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Detecting the hybridization probes bound to the filter. A variation of this procedure is a reverse Northern blot, in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from the tissue and labeled.

Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip, kit and test paper

The invention provides products for detecting the expression level of RP11-626H12.2 gene, including (but not limited to) chips, kits and test paper. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown by RP11-626H 12.2.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

"Probe" refers to a nucleic acid fragment as short as a few to as long as several hundred bases, such as RNA or DNA, that can establish specific binding to mRNA and can determine the presence of a particular mRNA by the action of a maintenance label (Labeling). The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA (single stranded DNA) probe, a double-stranded DNA (double stranded DNA) probe, an RNA probe, or the like. In the present invention, the prognosis of gastric cancer can be predicted by hybridization using the labeled polynucleotide of the present invention and a complementary probe, and by the presence or absence of hybridization. The appropriate choice of probes and hybridization conditions can be modified based on what is known in the art.

"hybridization" or "nucleic acid hybridization" or "hybridization" generally refers to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences that, under the appropriate conditions, will form a thermodynamically stable double-stranded structure. The term "hybridization" as used herein may refer to hybridization under stringent or non-stringent conditions. The setting of the conditions is within the skill of the person skilled in the art and can be determined according to the experimental protocols described in the art.

The term "hybridizing sequence" preferably refers to a sequence showing a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, particularly preferably at least 80%, more particularly preferably at least 90%, more particularly preferably at least 95%, and most preferably at least 97% identity. In the case of hybridization to nitrocellulose filters (or other such supports such as nylon), such as the well-known Southern blotting procedure, nitrocellulose filters can be incubated with labeled probes in overnight solutions containing high salt (6 XSSC or 5 XSSPE), 5 XDenhardt's solution, 0.5% SDS and 100. mu.g/ml denatured carrier DNA (e.g., salmon sperm DNA) at temperatures representing the conditions of desired stringency (high stringency 60-65 ℃, medium stringency 50-60 ℃, low stringency 40-45 ℃). Non-specifically bound probes can be detected by binding in 0.2 XSSC/0.1% SDS at a temperature selected according to the desired stringency: the filter was eluted from the wash several times at room temperature (low stringency), 42 ℃ (medium stringency) or 65 ℃ (high stringency). The salt and SDS concentrations of the wash solution may also be adjusted to suit the desired stringency. The temperature and salt concentration selected are based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions for hybridization and washing may be varied by those skilled in the art according to well-known methods, preferably using stringent conditions. Other protocols utilizing different annealing and washing solutions or commercially available hybridization kits (e.g., ExpressHybTM from BD Biosciences Clonetech) may also be used, as is well known in the art. It is well known that the length of the probe and the composition of the nucleic acid to be determined determine other parameters of the hybridization conditions. It is noted that variations of the above conditions can be achieved by the addition and/or substitution of alternative blocking reagents for suppressing background in hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA and commercially available proprietary formulations. Due to compatibility issues, the addition of specific blocking reagents may require modification of the hybridization conditions described above. Hybrid nucleic acid molecules also include fragments of the above molecules. In addition, nucleic acid molecules that hybridize to any of the above-described nucleic acid molecules also include complementary fragments, derivatives, and allelic variants of these molecules. In addition, a hybridization complex refers to a complex between two nucleic acid sequences that relies on the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. Two complementary nucleic acid sequences form hydrogen bonds in an antiparallel configuration. Hybridization complexes can be formed in solution (e.g., Cot or Rot assays), or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., a membrane, filter, chip, pin, or slide that has, for example, immobilized cells).

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

The invention provides a kit which can be used for detecting the expression of RP11-626H 12.2. The kit comprises a specific primer pair for amplifying RP11-626H 12.2; a standard DNA template; and (3) PCR reaction liquid. In a preferred embodiment, the specific primer pair comprises an upstream primer and a downstream primer, and the sequences are shown as SEQ ID NO. 1-2.

As a more preferable embodiment, the kit is a fluorescent quantitative PCR detection kit, and the primer is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes and composite probes.

In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.

In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg²⁺The fluorescent dye is SYBR Green II, and the Taq enzyme is hot start enzyme.

The invention provides test paper which can be used for detecting the expression of RP11-626H 12.2; the test paper comprises a probe which specifically recognizes RP11-626H12.2 or a primer pair which specifically amplifies RP11-626H 12.2.

In a preferred embodiment, the test strip further comprises a fibrous membrane, including but not limited to a nitrocellulose membrane or a nylon membrane.

In a more preferred embodiment, the fiber membrane is also provided with a detection line and a quality control line.

Inhibitors and pharmaceutical compositions

Based on the findings of the present invention, the present invention provides a pharmaceutical composition comprising an inhibitor of RP11-626H 12.2.

The inhibitor of RP11-626H12.2 refers to any substance capable of reducing the level of RP11-626H12.2 gene, and the substances can be used in the invention as substances which are useful for down-regulating the expression of RP11-626H12.2 gene, thereby being used for treating gastric cancer. For example, the inhibitor of the present invention can be an interfering molecule which uses the RP11-626H12.2 gene as a target sequence and can inhibit the RP11-626H12.2 gene, and comprises: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

The pharmaceutical composition comprises an effective amount of an RP11-626H12.2 inhibitor, and/or other medicines compatible with the inhibitor, and a pharmaceutically acceptable carrier and/or auxiliary materials.

The pharmaceutically acceptable carrier may be one or more and includes, but is not limited to, binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants and/or time delay agents (timedelayagent).

Drug screening

The invention provides a method for screening a medicine for treating gastric cancer, which comprises the following steps:

in the experimental group, adding a compound to be tested into a culture system, and measuring the expression level of RP11-626H 12.2; in a control group, no test compound is added into the same culture system, and the expression level of RP11-626H12.2 is measured; wherein, if the expression level of RP11-626H12.2 in the experimental group is less than that in the control group, the substance to be screened is a candidate substance of RP11-626H 12.2.

In the present invention, the method further comprises: the candidate substance obtained in the above step is further tested for its effect of inhibiting gastric cancer, and if the test compound has a significant inhibitory effect on gastric cancer, the compound is a candidate substance for treating gastric cancer.

Such culture systems include, but are not limited to, cell systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems (e.g., animal models, preferably non-human mammalian animal models, such as mice, rabbits, sheep, monkeys, etc.), and the like.

When a compound isolated by the screening method of the present invention is administered as a drug to a human or other mammal, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees, the isolated compound can be administered directly or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug may be orally administered as sugar-coated tablets, capsules, elixirs and microcapsules, as desired; or as a sterile solution or suspension in water or any other pharmaceutically acceptable liquid for parenteral administration in the form of an injection. For example, the compounds may be mixed together in unit dosage forms (unit dose) as required for generally accepted modes of pharmaceutical administration, with pharmaceutically acceptable carriers or media including, but not limited to, sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients (exipient), vehicles (vehicle), preservatives, binders and the like. Depending on the content of the active ingredient in these preparations, an appropriate amount to be administered within a prescribed range can be obtained.

Statistical analysis

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of Gene markers associated with gastric cancer

1. The transcriptome data of gastric adenocarcinoma patients, including 375 tumor cases, were downloaded in the TCGA database (https:// cancer. 35 cases in normal.

2. Transcriptome data integration analysis

1) Pretreatment of

After deleting the non-detectable lncRNA (i.e., the number of samples with the read counts of the lncRNA being 0 in case is more than 20% of the total case sample amount or the number of samples with the read counts of the lncRNA being 0 in normal is more than 20% of the total normal sample amount), 4610 lncRNA were used for differential expression analysis. And Principal Component Analysis (PCA) was performed on these lncRNA.

2) Differential expression analysis

Differential expression analysis was performed using DESeq2 in the R-3.3.3 tool, differential expression lncRNA screening criteria: FDR <0.05, abs (log2FC) > 2.

3) ROC analysis

An ROC curve is drawn for the differentially expressed genes, the area under the curve (AUC) is calculated to evaluate the diagnostic value of the differentially expressed genes for gastric adenocarcinoma, the AUC and the 95% confidence interval thereof are evaluated through MedCalc, and the differentially expressed genes can be divided into no prediction performance (AUC is less than 0.5), low prediction performance (AUC is more than or equal to 0.5 and less than or equal to 0.7), moderate prediction performance (AUC is more than or equal to 0.7 and less than or equal to 0.9) and high prediction performance (AUC is more than or equal to 0.9 and less than or equal to 1) according to the difference of the AUC values.

3. Results

The data analysis result shows that the expression of RP11-626H12.2 is up-regulated in gastric adenocarcinoma patients, ROC curve analysis is carried out on RP11-626H12.2 (figure 1), the curve area under the line is 0.9039, the specificity at the optimal critical value is 0.844, the sensitivity is 0.8455, the gene has high prediction performance, and RP11-626H12.2 can be applied to diagnosis of gastric adenocarcinoma.

Example 2 QPCR sequencing validation of differential expression of the RP11-626H12.2 Gene

1. Large sample QPCR validation of differential expression of the RP11-626H12.2 gene was performed. Cancer tissues and corresponding paracancerous tissues were collected from 30 patients with gastric adenocarcinoma.

2. RNA extraction

The specific operation of the Takara RNA extraction kit (Code NO.9767) for extracting RNA from tissues is detailed in the instruction.

3、QPCR

Primers were designed based on the gene sequences of RP11-626H12.2 and GADPH, and the primer sequences are shown in Table 1.

TABLE 1 amplification primer sequences

TaKaRa One Step TB Green^TM Prime Script^TMThe RT-PCR kit (Code No. RR066A) was used for PCR reaction, and the reaction system and reaction conditions are shown in Table 2. In the Thermal Cycler

PCR amplification is carried out on the Time System amplification instrument, after the reaction is finished, the amplification curve and the dissolution curve of Real Time PCR are confirmed, and relative quantification is carried out by the delta CT method.

TABLE 2 QPCR reaction System and reaction conditions

4. Results

The QPCR results are shown in fig. 2, compared with normal tissues, RP11-626H12.2 is up-regulated in gastric adenocarcinoma tissues, the difference is statistically significant (P <0.05), consistent with the high throughput sequencing results, suggesting that whether the subject has gastric adenocarcinoma can be judged by detecting the level of RP11-626H12.2, when the level of RP11-626H12.2 is significantly increased, the subject has gastric adenocarcinoma or is at risk of having gastric adenocarcinoma, shRNA, siRNA and the like that reduce the level of RP11-626H12.2 can be designed to treat gastric adenocarcinoma through the relationship between RP11-626H12.2 and gastric adenocarcinoma.

Example 3 RP11-626H12.2 Regulation of gastric cancer cell proliferation and migration

1. Cell culture

Taking out the frozen gastric adenocarcinoma cells SGC-7901 from liquid nitrogen, placing in a 37 ℃ water bath for rapid thawing, transferring the thawed cells into a centrifuge tube containing a culture medium, centrifuging for 3min, discarding the supernatant, suspending the cells uniformly by a 2m1 complete culture medium, transferring into a culture bottle, and culturing at 37 ℃ with 5% CO₂Culturing in the incubator. After 24h, the cells were observed for growth state and morphology, and the culture medium was replaced with the cells. After 3 passages, cells grown in a logarithmic state were selected for subsequent experiments.

2. Cell transfection

The experiment was divided into three groups, control (SGC-7901), negative control (siRNA-NC) and experimental (siRNA-RP11-626H 12.2). Grouping according to experiments, after counting cells, inoculating the cells into a six-hole plate, culturing overnight, enabling the cell density to reach 70%, preparing a solution 1 by using Lipofectamine3000 and DMEM in proportion, preparing a solution 2 by using siRNA or NC and DMEM in proportion, mixing the siRNA or NC and the DMEM, incubating for 15min at room temperature, adding into corresponding cell culture holes, and adding a complete culture medium for culturing after 5 h.

Wherein, the general siRNA-NC and siRNA-RP11-626H12.2 used in the application are purchased from Shanghai Ji code pharmaceutical technology Co., Ltd, and the sequences of the siRNA-RP11-626H12.2 for silencing RP11-626H12.2 are shown in Table 3.

TABLE 3 sequences of siRNA

3、qRT-PCR

Total cellular RNA was extracted using Trizol method, reverse transcription and real-time quantitative PCR detection were performed as in example 2.

4. CCK-8 cell proliferation assay

Cells in log phase were seeded at 4000 cells per well in 96-well plates and transfected when the 96-well plate cells reached 80% confluence. When the transfection time reaches 24 hours, the time point is taken as the starting point of detection and is marked as 0 hour, after 48 hours of culture, the culture solution is sucked, 100 mul of complete culture medium and 10 mul of CCK-8 reagent are added into each hole, after 1 hour of incubator incubation, the light intensity of the microplate reader is adjusted to 450nm, and the light absorption value of each group is measured.

5. Transwell cell migration experiment

Culturing each group of cells to 90% confluence, digesting with pancreatin, and centrifuging to remove the supernatant. And (5) uniformly blowing the serum-free culture medium, and counting cells. 200. mu.l of medium, 5X 10 per chamber⁴Cell standards were added to the corresponding chambers. And adding 750 mu l of culture medium containing 10% FBS into the lower chamber, putting the lower chamber into an incubator for culturing for 24 hours, and observing the adherence condition of the cells in the lower chamber by a microscope. The chamber was removed, the medium was decanted, the cells were fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet for 20 min. Cell counts were performed under microscope and analyzed statistically.

6. Statistical analysis

The experiments were performed in 3 replicates, and the results were expressed as mean ± sd, and the difference between the two was considered statistically significant when P <0.05 using the t-test.

7. Results

The results of qRT-PCR are shown in Table 4, compared with the control group, the relative expression level of RP11-626H12.2 of the siRNA-RP11-626H12.2 group is obviously reduced (P < 0.05); the relative expression level of RP11-626H12.2 in the negative control group was not significantly changed (P > 0.05).

TABLE 4 RP11-626H12.2 relative expression

The CCK-8 cell proliferation experiment result is shown in Table 5, compared with the negative control group, the cell proliferation of the experimental group is obviously inhibited, which shows that the expression of RP11-626H12.2 is reduced, the growth of cancer cells can be inhibited, and therefore, a new means is provided for the treatment of the development of the gastric cancer.

TABLE 5 OD values

As shown in table 6, compared with the negative control group, the cell migration number of the experimental group is significantly reduced, which indicates that the reduction of the expression of RP11-626H12.2 can inhibit the migration of cancer cells, thereby providing a new strategy for the treatment of gastric cancer metastasis.

TABLE 6 number of migrating cells

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> ninth and sixth 0 Hospital of the United service guarantee army of the liberation force of people in China

<120> molecular marker for developing diagnosis and treatment product for gastric adenocarcinoma

<150> 201910480351X

<151> 2019-06-04

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aacgatgact acgaatgaga 20

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttgggcaaca gaatgaga 18

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aatcccatca ccatcttcca g 21

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagccccagc cttctccat 19

<210> 5

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ucauucguag ucaucguucg c 21

<210> 6

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaacgaugac uacgaaugag a 21

Claims (10)

1. A reagent capable of detecting the level of RP11-626H12.2 gene in a sample.

2. The reagent according to claim 1, comprising:

a probe specifically recognizing RP11-626H 12.2; or

And (3) primers for specifically amplifying RP11-626H 12.2.

3. The reagent according to claim 2, wherein the primer sequence for specifically amplifying RP11-626H12.2 is shown as SEQ ID No. 1-2.

4. A kit comprising the reagent of any one of claims 1 to 3.

5. A chip comprising the reagent according to any one of claims 1 to 3.

6. A test strip comprising the reagent according to any one of claims 1 to 3.

7. A pharmaceutical composition comprising an inhibitor of RP11-626H 12.2.

8. The pharmaceutical composition of claim 7, wherein the inhibitor is an agent that reduces the expression level of RP11-626H 12.2.

9. A method of screening for a candidate substance for treating gastric cancer, the method comprising:

treating a culture system expressing or containing an RP11-626H12.2 gene with a substance to be screened; and

detecting the expression of RP11-626H12.2 gene in the system;

wherein, if the substance to be screened can inhibit the level of the RP11-626H12.2 gene, the substance to be screened is a candidate substance for treating gastric cancer.

10. Use according to any one of the following:

a. use of an agent according to any one of claims 1 to 3 for the manufacture of a product for the diagnosis of gastric adenocarcinoma;

b. use of the kit of claim 4 for the preparation of a product for the diagnosis of gastric adenocarcinoma;

c. use of the chip of claim 5 for the preparation of a product for diagnosing gastric adenocarcinoma;

d. use of a strip according to claim 6 in the manufacture of a product for diagnosing gastric adenocarcinoma;

application of RP11-626H12.2 in constructing a calculation model for predicting gastric adenocarcinoma;

f. use of a pharmaceutical composition according to claim 7 or 8 for the preparation of a product for the treatment of gastric adenocarcinoma.

Images